High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells.
The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 microM tetrabenazine and 10 microM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.
Sievert
M K
MK
Department of Pharmacology, University of Wisconsin Medical School, Madison, WI 53706, USA.
Thiriot
D S
DS
Edwards
R H
RH
Ruoho
A E
AE
D000818:Animals; D016367:Baculoviridae; D001692:Biological Transport; D002417:Cattle; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D010777:Photochemistry; D051381:Rats; D018411:Spodoptera; D013572:Synaptic Vesicles / Q000378:metabolism*; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000378:metabolism; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; C493454:Slc18a2 protein, rat; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; C032811:dihydrotetrabenazine; D013747:Tetrabenazine